Although it is clear that solute transport across the cell membrane is an important determinant in the rate of cellular metabolism, little is known about the molecular details of transport processes. This projjct is an investigation of active transport in bacteria and our objective is to understand the mechanism of solute uptake at a molecular level. Our specific aims are to find out the following: (1) What are the properties of carrier proteins that catalyze translocation of hydrophilic substrates across the hydrophobic core of the plasma membrane? (2) What is the role of ion pumps and/or a transmembrane potential in mediating uphill movement of solutes? (3) What is the nature of membrane phospholipid involvement in carrier function and in the coupling of metabolic energy to active transport. Our approach to these problems involves study of respiration-coupled transport in membrane vesicles insolated from Azotobacter vinelandil, a strictly aerobic bacterium. We plan to develop methods for isolation and study of the calcium transport protein from these vesicles. In addition, we will examine and compare the energy requirements for the uptake of glucose, calcium, acetate, and triphenylmethylphosphonium ion. Finally, the effect of phospholipase treatment of vesicles on binding and uptake of these slutes will be studied.